Cyclic nucleotide-gated (CNG) ion channels are key players in the early stages of vision. They are participants in an enzyme cascade in rods and cones that converts the energy of absorbed light into an electrochemical signal to be relayed to the brain. The channels are opened by the direct binding of cyclic guanosine monophosphate (cGMP), whose free concentration is higher in the dark than in the light. Closure of the channels in the light causes a change in membrane potential that reduces the release of neurotransmitter onto second order neurons. The apparent affinity of the CNG channels for cGMP has been found to be modulated by cytosolic and membrane factors, including Ca2+/calmodulin, phosphatases and diacylglycerol. There is also evidence that the concentration and/or effectiveness of some modulatory factors depends on the amount of light absorbed. Functional modulation of the CNG channels has potential significance in two ways: 1) it may be involved in some aspect of normal visual transduction, such as in light or dark adaptation; and 2) its pharmacologic or genetic manipulation may be useful in treating some forms of retinal degeneration in which either the channel or the cGMP concentration is abnormal. CNG channels are composed of at least two kinds of subunits, alpha and beta, both of which contain cyclic nucleotide binding sites. Ca2+/calmodulin has been found to act only on the beta subunit, but it is not known which subunit(s) are controlled by the other modulators. The long-range goals of this project are to understand how rod and cone CNG channels work and how they participate and are controlled in visual transduction. The specific aims are to use electrophysiology and molecular biology techniques to study functional modulation of rod CNG channels by Ca2+/calmodulin and phosphorylation. Initially, modulation by phosphorylation will be studied in native channels from amphibian rods to determine the roles of specific phosphatases and kinases. The Xenopus oocyte expression system and patch clamp studies will allow functional analysis of wild-type cloned bovine channels as well as mutants designed to dissect the molecular mechanisms of the two forms of modulation.